Skin pigment irregularities are common across ethnic and racial groups and are often considered cosmetically disfiguring. Disorders of pigment production and distribution occur as a function of intensity and duration of UV radiation exposure, life style habits, chronological age, endocrine functioning and disease state and are found ubiquitously in older populations. Hence there is a widespread demand for skin pigment modifying, skin lightening and skin tone enhancing products for the cosmetic market.
The color of normal human skin is due primarily to varying amounts and distribution of melanin, hemoglobin, and carotenoids. Of these pigments, melanin is of primary significance to cosmetic skin treatment protocols. Melanin is produced by specialized cells in the skin called melanocytes through a complicated series of chemical and enzymatic reactions, mainly involving the copper and manganese containing enzyme tyrosinase. Once synthesized, the melanin granules are packaged into melanosomes and transferred via the cellular dendrites (extensions) of the melanocyte to the surrounding keratinocytes, the most abundant cell type in the epidermis. The rate of melanin synthesis, and the subsequent transfer of melanin by melanocytes via their dendrites, appears to be influenced by ultraviolet light exposure. Melanosomes transferred to the outer layer of the skin are responsible for the darkening of the skin, with the degree of darkening being associated with skin type, sun exposure, and/or certain dermatological conditions.
Two types of melanin are present in human skin: (1) eumelanin, which is the dark brown-black pigment found in most skin, hair, and eyes, and whose production is stimulated by exposure to ultraviolet light, and (2) pheomelanin, which is a yellow-orange pigment found mainly in the skin of very fair-skinned people, particularly those with red hair. The perceived color of skin is determined by the ratio of eumelanins to pheomelanins, and to a smaller extent on blood within the dermis.
The pigmentation pathway has been elucidated in detail. Summarily, melanin forms through a series of oxidative reactions involving the amino acid tyrosine in the presence of the enzyme tyrosinase. Tyrosinase converts tyrosine to dihydroxyphenylalanine (DOPA) and then to dopaquinone. Subsequently, dopaquinone is converted to dopachrome through auto-oxidation, and finally to dihydroxyindole or dihydroxyindole-2-carboxylic acid (DHICA), which polymerize to form eumelanin. The latter reactions occur in the presence of dopachrome tautomerase and DHICA oxidase. In the presence of sulfur-containing cysteine or glutathione, dopaquinone is converted to cysteinyl DOPA or glutathione DOPA; subsequently, pheomelanin is formed.
A variety of skin hyperpigmentation disorders are known and etiology is diverse, overlapping in many cases, and often not fully understood. For example, melanosis or melasma is a condition characterized by the development of sharply demarcated blotchy, brown spots usually in a symmetric distribution over the cheeks, forehead, and sometimes on the upper lip and neck. This condition frequently occurs during pregnancy (melasma gravidarum or “mask of pregnancy”), and at menopause. Also, this condition is frequently found among those taking oral contraceptives, and is occasionally found among nonpregnant women who are not taking oral contraceptives, and sometimes among men. A pattern of similar facial hyperpigmentation is associated with a chronic liver disease called chloasma. A common condition associated with aging skin is the development of dark spots sometimes referred to as “age spots” or “liver spots.” Other forms of hyperpigmentation can be caused by UV irradiation, in particular UVB radiation which up-regulates the production of tyrosinase resulting in skin “tanning,” or may result from a genetic predisposition for the condition, or may come about in association with a skin inflammatory event or during the course of wound healing.
Vitiligo is a form of hypopigmentation in which cutaneous melanocytes are either ablated or fail to produce sufficient pigment. Ideally treatment would restore lost pigmentation in vitiligo-affected skin, but this approach has met with little success via topical interventions and formulations. Although cosmetic camouflage with dihydroxyacetone sunless-tanning lotions provides some darkening of hypo-pigmented areas, it also tends to darken surrounding normal skin, substantially maintaining the undesirable contrast. Hence, a more favored cosmetic approach is to reduce the normal pigmentation of the unaffected skin to reduce contrast and produce a tone evening effect.
Several proven targets for pigmentation control are known, but these have generally been derived from an understanding of the pigmentation process. Hydroquinone (parahydroxy-benzene), for example, is a widely used skin lightening agent that is known to provide a satisfactory cosmetic result, however its use strictly for cosmetic purposes is discouraged due to its association with a variety of disorders, including diabetes, hypertension, ochronosis, periorbitary dyschromia, infectious dermatosis, contact eczema, extended dermatophytosis, and necrotizing cellulites (see, e.g., Raynaud E. et al., Ann Dermatol Venereol 128(6-7):720-724, 2001). Hydroquinone has also shown genotoxic and mutagenic activities (see, e.g., Jagetia G. C. et al, Toxicol Lett 121(1):15-20, 2001). Due to concerns over toxicity and carcinogenic effects, the United States limits treatment solutions to a 2% or lower concentration and the FDA has proposed a ban on all over-the-counter preparations, while hydroquinone is currently banned in Europe as a skin lightening or depigmenting agent.
Kojic acid, Azelaic acid and certain-hydroxy acids such as glycolic acid, have demonstrated skin-lightening effects, but reports of localized irritation and inflammation are common. The prenylated flavonol artocarpin has shown some efficacy for skin-lightening in the context of ultraviolet-induced skin pigmentation (Shimizu K. et al., Planta Med 68(1):79-81, 2002).
Recently, a more detailed genomic and proteomic understanding of melanogenesis, the melanocyte, melanocyte-keratinocyte interaction, and the melanocyte-fibroblast interaction has revealed potentially hundreds of proteins and other effectors involved in the pigmentation process and in the etiology of hyperpigmentation disorders, which may provide additional targets. There is a need in the cosmetic arts both for generating potential skin lightening agents and for effective and efficient screening methods for identifying putative skin active agents with efficacy and safety in the cosmetic treatment of hyperpigmentation and pigmentation disorders.
Traditionally scientists have focused on the development and provision of safe and effective topical compositions formulated to lighten skin and such an approach has been useful for treating localized epidermal hyperpigmentation and for masking areas of skin hypopigmentation. There remains a need, however, for safe and effective agents capable of delivery through topical application to reduce the degree of skin pigmentation in both contexts.
Skin pigmentation and the broader cosmetic concept of skin tone, are therefore highly complex conditions with multiple and overlapping etiologies, which manifest in part as a function of individual predisposition, and which therefore pose a significant treatment challenge. There is a need in the art for methods of identifying potential skin pigment modifying agents, and in particular skin-lightening agents, and for evaluating the efficacy of putative skin active agents using screening methods that are substantially independent of mechanism of action or etiology of the pigment condition. The present investigators therefore undertook an investigation into the application of a relatively new technology known as “connectivity mapping” to the search for new skin-active agents with efficacy in the treatment of hyperpigmentation disorders and related skin conditions.
Connectivity mapping is a well-known hypothesis generating and testing tool having successful application in the fields of operations research, telecommunications, and more recently in pharmaceutical drug discovery. The undertaking and completion of the Human Genome Project, and the parallel development of very high throughput high-density DNA microarray technologies enabling rapid and simultaneous quantization of cellular mRNA expression levels, resulted in the generation of an enormous genetic database. At the same time, the search for new pharmaceutical actives via in silico methods such as molecular modeling and docking studies stimulated the generation of vast libraries of potential small molecule actives. The amount of information linking disease to genetic profile, genetic profile to drugs, and disease to drugs grew exponentially, and application of connectivity mapping as a hypothesis testing tool in the medicinal sciences ripened.
The general notion that functionality could be accurately determined for previously uncharacterized genes, and that potential targets of drug agents could be identified by mapping connections in a data base of gene expression profiles for drug-treated cells, was spearheaded in 2000 with publication of a seminal paper by T. R. Hughes et al. [“Functional discovery via a compendium of expression profiles” Cell 102, 109-126 (2000)], followed shortly thereafter with the launch of The Connectivity Map (-map Project by Justin Lamb and researchers at MIT (“Connectivity Map: Gene Expression Signatures to Connect Small Molecules, Genes, and Disease”, Science, Vol 313, 2006.) In 2006, Lamb's group began publishing a detailed synopsis of the mechanics of C-map construction and installments of the reference collection of gene expression profiles used to create the first generation C-map and the initiation of an on-going large scale community C-map project, which is available under the “supporting materials” hyperlink at http://www.sciencemag.org/content/313/5795/1929/suppl/DC1.
The basic paradigm of predicting novel relationships between disease, disease phenotype, and drugs employed to modify the disease phenotype, by comparison to known relationships has been practiced for centuries as an intuitive science by medical clinicians. Modern connectivity mapping, with its rigorous mathematical underpinnings and aided by modern computational power, has resulted in confirmed medical successes with identification of new agents for the treatment of various diseases including cancer. Nonetheless, certain limiting presumptions challenge application of C-map with respect to diseases of polygenic origin or syndromic conditions characterized by diverse and often apparently unrelated cellular phenotypic manifestations. According to Lamb, the challenge to constructing a useful C-map is in the selection of input reference data which permit generation of clinically salient and useful output upon query. For the drug-related C-map of Lamb, strong associations comprise the reference associations, and strong associations are the desired output identified as hits.
Noting the benefit of high-throughput, high density profiling platforms which permit automated amplification, labeling hybridization and scanning of 96 samples in parallel a day, Lamb nonetheless cautioned: “[e]ven this much firepower is insufficient to enable the analysis of every one of the estimated 200 different cell types exposed to every known perturbagen at every possible concentration for every possible duration . . . compromises are therefore required” (page 54, column 3, last paragraph). Lamb, however, took the position that cell type did not ultimately matter, and confined his C-map to data from a very small number of established cell lines out of efficiency and standardization concerns. Theoretically this leads to heightened potential for in vitro to in vivo mismatch, and limits output information to the context of a particular cell line. If one accepts the Lamb precept that cell line does not matter then this limitation may be benign.
However, agents suitable as pharmaceutical agents and agents suitable as cosmetic agents are categorically distinct, with the former defining agents selected for specificity and which are intended to have measurable effects on structure and function of the body, while the latter are selected for effect on appearance and may not affect structure and function of the body to a measurable degree. Cosmetic agents tend to be substantially non-specific with respect to effect on cellular phenotype, and administration to the body is generally limited to application on or close to the body surface.
In constructing C-maps relating to pharmaceutical agents, Lamb stresses that particular difficulty may be encountered if reference connections are extremely sensitive and at the same time difficult to detect (weak), and Lamb adopted compromises aimed at minimizing numerous, diffuse associations. Since the regulatory scheme for drug products requires high degrees of specificity between a purported drug agent and disease state, and modulation of disease by impacting a single protein with a minimum of tangential associations is desired in development of pharmaceutical actives, the Lamb C-map is well-suited for screening for potential pharmaceutical agents despite the Lamb compromises.
The connectivity mapping protocols of Lamb would not be predicted, however, to have utility for hypothesis testing/generating in the field of cosmetics or for a primarily cosmetic disorder where symptoms may be diffuse, systemic and relatively mild. In complete contravention of the goal of pharmaceutical active discovery, cosmetic formulators seek agents or compositions of agents capable of modulating multiple targets and having effects across complex phenotypes and conditions. Further, the phenotypic impact of a cosmetic agent must be relatively low by definition, so that the agent avoids being subject to the regulatory scheme for pharmaceutical actives. Nonetheless, the impact must be perceptible to the consumer and preferably empirically confirmable by scientific methods. Gene transcription/expression profiles for cosmetic conditions are generally diffuse, comprising many genes with low to moderate fold differentials. Cosmetic agents, therefore, provide more diverse and less acute effects on cellular phenotype and generate the sort of associations expressly taught by Lamb as unsuitable for generating connectivity maps useful for confident hypothesis testing.
Successful identification of skin lightening agents has proven to be difficult due to the multi-cellular, multi-factorial processes involved in etiology of the hyperpigmentation condition itself. Conventional in vitro studies of biological responses to potential skin-lightening agents can be hindered by the complex or weakly detectable responses typically induced and/or caused by the putative skin active or potential skin active agents. Such weak responses arise, in part, due to the great number of genes and gene products involved, and the fact that skin-active and cosmetic agents may affect multiple genes in multiple ways. Moreover, the degree of bioactivity of cosmetic agents may differ for each gene and be difficult to quantify.
The value of a connectivity map approach to discover functional connections among cosmetic phenotypes such as hyperpigmented skin, gene expression perturbation, and cosmetic agent action is counter-indicated by the progenors of the drug-based C-map. The relevant phenotypes are very complex, the genetic perturbations are numerous and weak, and cosmetic agent action is likewise diffuse and by definition, relatively weak. It is unclear whether statistically valid data may be generated from cosmetic C-maps and it is further unclear whether a cell line exists which may provide salient or detectable cosmetic data.